Ulcerative colitis, microbiota and prebiotics

Ulcerative colitis, microbiota and prebiotics

Pavel Nesmiyanov, MD, PhD

Abstract

Inflammatory bowel diseases are characterized by complex interactions of the immune system and gut microbiota. With the recent technological advances, it became possible to provide new insights into disease pathogenesis. Accumulating evidence suggests the utility of microbiota in diagnostics, prognostics, and therapeutics in inflammatory bowel diseases. In this review we focus on alterations of microbiota and immune system in ulcerative colitis and issues on the use of prebiotics and novel prebiotic formulations.

Keywords: ulcerative colitis, microbiota, prebiotics

Figure 1. Pathogenetic networks in ulcerative colitis

Background

Ulcerative colitis (UC) is a chronic and relapsing inflammatory bowel disease (IBD) restricted to the large intestine, in contrast to Crohn’s disease (CD) which may involve any part of the gut. Despite of unknown etiology of UC, many factors are described to contribute to the disease: genetic background, immune system reactions, environmental factors, NSAIDs use, low levels of antioxidants, psychological stress factors, and dairy products consumption[1–3]. Among all these factors, immune reactions (both genetically and environmentally defined), play a major role, to our opinion.

Immune dysregulation has long been suspected to play a role in the course of inflammatory bowel disease with earliest research that dates back to 1970s[4, 5]. UC is characterized by a plethora of immunological changes, including accumulation of neutrophils and activated cytotoxic T cells in the lamina propria of the affected colonic region, increased B cell count with corresponding IgG and IgE elevation, some of which are specific to colonic antigens [57]. The presence of antineutrophil cytoplasmic antibodies and anti-Saccharomyces cerevisae antibiodies is a common feature in UC[6–10]. Interestingly, appendectomy leads to a lower incidence of UC[11, 12]: one hypothesis is that alterations in mucosal immune responses leading to appendicitis or resulting from appendectomy may negatively affect the pathogenetic mechanisms of UC. Abnormal function of immunoregulatory cytokines, such as IFN-γ, TNF-α in animal models of UC is well documented. Microscopic changes include signs of chronic inflammation, crypt branching and villous atrophy as well as formation of inflammatory polyps. Sufficient evidence have been provided recently on the involvement of gut microbiota in the pathophysiology of UC. Dysregulation of the immune system, it’s inappropriate immune responses to intestinal microorganisms and their products, continuous inflammation have profound effects on gut microbiota. Vice versa, changes in microbiota play a role in development of inflammatory bowel disease, as discussed below.

Ulcerative colitis and microbiota

New insights into the composition of gut microbiota in health and disease have been provided with the development of next-generation sequencing techniques. Generally, gut microorganisms are seen as a system of two phylums: Firmicutes (49-76%) and Bacteroidetes (16-23%)[1] although members of at least 10 different phyla (Proteobacteria, Actinobacteria etc.) are functionally important[13].

Mucosa-associated microorganisms are increased in IBD, and are highly coated in sIgA[14] and probably are more relevant in UC than that found in fecal samples[15, 16]; an imaging study using approaches that carefully preserve the structure of faeces also identified discrete patches; individual groups of bacteria were found to spatially vary in abundance from undetectable to saturating levels[17].

Changes of microbiota in UC are summarized in Table 1. Usually, healthy state is characterized by a relative temporal stability of microbiota, while UC patients have reduced diversity of gut microorganisms [1, 18], largely due to a decrease in the diversity of Firmicutes and an increase of Proteobacteria. Though the ratio of Firmicutes:Bacterioidetes is not the same even in healthy individuals, even a single species depletion, such as Faecalibacteriumprausnitzii (Firmicutes phylum) can be associated with IBD [19, 20]. Discrepant results are found in the studies on E.coli,Bifidobacteria species, Lactobacillus species, Enterobacteriaceae and Bacteroides [16, 21, 22]. The common feature in UC patients is that the composition of gut microbiota differs between active and remission stages and even unstable in quiescent stage [23]. Exacerbations of UC is preceded by decrease in so-called “anti-inflammatory” anaerobes, e.g Bacteroides, Escherichia, Eubacterium, Lactobacillus, and Ruminococcus and diminution in the diversity of the gut microbiota [24].

In other reports a localized dysbiosis found. For example, inflamed and non-inflamed sites of the mucosa are substantially different in the prevalence of Lactobacilli and the Clostridium leptum subgroup and that this may be related to UC [25, 26]. Proteobacteria and Firmicutes were more frequently observed in the inflamed UC mucosa [27]. However, recent research has found no correlation between regional inflammation and a breakdown in this spatial differentiation or bacterial diversity [28]. Dysbiosis not only affects bacterial species. It has been shown that disturbances in microbiota promote specific Candida and Cladosporium fungi [29] as well as virome components [30].

To summarize, bacterial dysbiosis has been proven to play an important role in UC pathogenesis. Mucosal dysbiosis may have diagnostic, prognostic, and therapeutic implications. Whether modification of microbiota is a cause or a consequence of intestinal inflammation in UC is still a matter of debate. Data on specific microbiota shifts also vary. More well-designed trials are required to show UC-specific changes. Studies should focus on mucosa-associated microbiota. The findings can be viewed in the context of microbiota-associated pathogenic network in UC comprising two perspectives: how UC affects gut microbiota and how microbiota affects UC.

Table 1. Summary of microbiota major changes in UC compared to healthy subjects

Microbiota-associated pathogenic networks in ulcerative colitis

Ulcerative colitis and associated immune changes induce gut dysbiosis

Though UC is a complex disease leading to many perturbations that may affects gut microbiota, the main focus can be put on two pathogenic pathways that influence the bacterial composition: first, morphological changes (tissue impairment), and second, immune dysregulation. Sufficient evidence also found on the on the selection of intestinal flora by dietary habits [31].

Tissue impairment as a factor of microbiota disbalance

Tremendous quantity of anaerobic bacteria in the distal small intestine and large intestine is a factor that predisposes to an inflammatory response in the case of any epithelial damage. Epithelium defects (spontaneous or post-NSAID) allow bacterial antigens to stimulate immune cells in the mucosa and to induce inflammation, as it was shown on mouse models [32]. In IBD, quantity of mucosal bacteria is increasing, which is correspondent with the finding that many of the UC susceptibility genes are involved in mucosal barrier consistency [16]. DSS-induced colitis in mice is also characterized by overall increase of mucosa-associated bacteria (consistent with the findings in human UC [33]) and suppression of mucosal Lactobacillus species at the same time [34], as well as reduced microbial diversity [35]. Another example is an increase in auxotrophic bacteria functions, including a decrease in amino acid biosynthesis, and an increase in amino acid transfer. These bacteria benefit from conditions where nutrients are released during inflamed tissue destruction and are easily accessible in the environment [99]. Furthermore, mucin-degrading bacteria are increased in IBD, for example Ruminococcus gnavus and Ruminococcus torques, which utilize mucin as an energy source and provide degraded mucins as nutrients for other bacteria [36]. However, mucin production in IBD is impaired due to Goblet cell defects. This allows us to propose that failure in immune inhibition of the bacteria, described below, rather than the increased source of digestible endogenous mucin, enhances the presence of mucosa-associated bacteria in IBD.

Immune dysregulation as a factor of microbiota disbalance

Aberrant immune response in UC is of great significance. For example, targeted knockout (KO) of IL-2 and IL-10 in rodents as well as HLA-B27 knockin leads to the development of IBD in the presence of a normal microflora but not in a germ-free animals [37]. Numerous murine strains develop IBD as a result of genetic manipulation or targeted knock out (KO) of genes that affect the mucosal immune system (T-bet, IL-2-KO, IL-2R-KO, IL-10-KO, TCR-KO, TGF-β-KO, SMAD 3-KO, MHC class II, WASP, A20, COX-1 and COX-2) or epithelial function (Gαi2-KO, XBP1) [37–47] which in turn modulate microbiota composition. Overexpression of genes, responsible for immune interaction with microbial antigens (eg, HLA-B27, N-cadherin, CD3e) or deficiency of regulatory cell types (eg, CD4+ CD45RBlo, CD25+ T-cells) that are involved in either lymphoid or epithelial cell homeostasis also leads to IBD-like condition [32, 47–51]. An interesting discover, connecting gut microbiota composition and immune function (T-cell dependent/independent IgA secretion, regulated by ILC-produced lymphotoxins), has been made recently by Nedospasov team [52].

Gut dysbiosis is a risk factor for the UC: pathogens and commensals

Gut dysbiosis can be directly responsible for initiating or promoting UC. Indirect confirmation of this statement is the fact that broad-spectrum antibiotics have been shown to reduce clinical signs of inflammation in UC [53–55]. At the same time antibiotic exposure during the first year of life has been identified as independent risk factors for IBD [56–58]. Altered microbiota precedes the onset of colitis mouse models [59]. Not to mention, recent studies have confirmed the immunoregulatory role of some gut bacteria, with certain species favoring the growth of protective or inflammatory T-cell subsets, such as Bacteroides fragilis (↑Treg), Clostridia and segmented filamentous bacteria (SFB) (↑Th17) [60–62]. Moreover, pathiological changes of the gut microbiota are associated with certain genotype (NOD2 and ATG18L alleles, which are two major CD susceptibility genes) in humans [63], which is consistent with the theory that genetic and environmental factors, rather than inflammation, induce dysbiotic changes.

Pathogens

Unfortunately, no research to date has either confirmed or denied the precise role of any particular organisms in IBD. However, several specific bacteria have been identified to be associated with UC. Documented Salmonella or Campylobacter gastroenteritis increased risk of developing IBD compared to a control group during the 15-year follow-up period [64, 65]. This finding is of great significance taking into account the prevalence of Salmonella carriage among asymptomatic children [66]. These bacteria induce inflammation by invading into intestinal epithelium, increasing permeability and causing cell damage and microvillus degradation [67]. It was also observed that Fusobacterium varium invades the inflamed mucosa in UC, inducing corresponding humoral response; its role in colitis induction has been confirmed in animal models [68–70]. In other studies, clostridial-specific T cells are present in mice with experimental colitis, and these T cells induce colitis when adoptively transferred into immunodeficient mice [71].

Commensals

Commensal bacteria are thought to be the primary trigger of intestinal inflammation in those who may be susceptible to UC; however, the relationship between UC pathogenesis and commensal bacteria is not completely understood. It is widely accepted that the increased exposure of mucosal T cells to bacteria results in chronic inflammation [72]. “Proinflammatory” bacteria — Campylobacter spp [73], E. coli [15, 74, 75], Enterohepatic Helicobacter [76], and Bacteroides ovatus [45] have been shown to induce the intestinal inflammation. In contrast, “anti-inflammatory” Pediococcus acidilactici and Lactobacillus spp are increased during remission of UC, probably stimulating Tregs and maintaining gut gomeostasis [77, 78]. A recent study reported that mucosal bacteria of UC patients failed to induce colitis in human microbiota‑associated mice but the same bacteria increased the susceptibility to DSS-induced colitis [79], thus supporting the multifactorial nature of UC. Analogues of SFB were observed to be prevalent in UC patients [80], in contrast to healthy controls.

A study, in which a single strain of bacteria was transferred into germ-free IL-10-KO mice, demonstrated that E. coli induced mild cecal inflammation and Enterococcus faecalis induced distal colitis [81]. These results show that alteration of the composition of the gut microbiota can cause distinct intestinal immune responses even in a genetically same host. Garrett et al. [38] reported that immunodeficient mice with both T-bet (Th1 signature transcription factor), and RAG (enzyme essential to the generation of mature B and T lymphocytes) knockout developed spontaneous UC-like colitis. Treatment with the broad spectrum antibiotics combination cured the mice of their colitis, as wells as selective treatment with metronidazole alone. What is more intriguing, co-housing wild-type mice with colitic Tbet/Rag double knockout animals led to development colitis in wild-type mice, raising the possibility that colitic gut microbiota is communicable.

The mechanisms of UC triggering by microbiota involve metabolic alterations. Gut microbiota products control functions of epithelium and energy balance. Analysis of gut microbiota metagenome showed a decrease in genes responsible for carbohydrate and amino acid metabolism and an increase in those in the oxidative stress pathway [82], suggesting that bacterial oxidative stress causes intestinal inflammation. Also, gut bacteria synthesize short-chain fatty acids (SCFA), such as butyrate and propionate. These SCFAs control the immune system by inducing the differentiation of colonic regulatory T cells [83–87]. Clostridia— and Bacteroides-derived butyrate is used by epithelium as an energy source for the production of mucin and AMPs [88]. Therefore, decreased concentrations of Clostridium groups IV and XIVa [89] could explain the observed decreased SCFA concentrations in fecal extracts of IBD patients [90]. Inflammatory processes in the intestine are promoted by decline in butyrate production. Consistently, one of the butyrate producers, F. prausnitzii, is decreased in IBD [16]. Overgrowth of sulfate-reducing bacteria (SRB) in UC has also been reported [91, 92]. SRB produce hydrogen sulfide, which blocks butyrate utilization by colonocytes [93] and can cause mucosal inflammation.

Thus, it seems that dysbalance of the gut commensal composition is involved in the pathophysiology of UC. However, studies in twins show inconsistent results when comparing healthy individuals and individuals with UC [94, 95]. Moreover, a decrease in F. prausnitzii was reported to be observed in both UC patients and their first-grade relatives [96]. Hence, we can’t exactly define the microbial profile associated with UC; the situation gets more complicated because many studies come from animal models – however, most mouse models (currently about 60 different models) of colitis do not fully recapitulate the pathophysiology of human UC [97]. For example, Akkermansia was found to be reduced in abundance in a human UC study [98], but was increased in the DSS-induced mouse model [99].

Treatment regimens affect gut microbiota in UC

There are many treatment options for the UC patients with emerging novel therapies such as fecal microbiota transplantation and Microbial Ecosystem Therapeutics. Approved medications for UC management are:

Any medication may affect the composition of gut microbiota. 5-ASA, for example, noticeably reduces the gut bacteria quantity [100]. However, there were no dramatic differences in fecal microbiota pattern between newly diagnosed, untreated patients with IBD and previously reported long-term (treated) patients [101]. Antibiotic treatment in IBD is associated with an increased risk of Clostridium dificille infection (CDI) which has worse outcomes in this patients [102, 103]. On the other hand, a study of patients with IBD found that antibiotics, after cessation of therapy, increase concentration of mucosal bacteria to 25-fold numbers compared to patients without antibiotic treatment [104].

Anti-TNF therapeutics dampens inflammation in IBD, at least in part, by modulating the gut microbiota toward eubiosis. The microbiome of treated individuals was characterized by reduced Enterobacteriaceae (specifically E. coli) and Ruminococcus, and increased proportions of Bacteroidetes and Firmicutes, restoring the microbiome to a composition more reflective of healthy individuals [105].

Important application of the microbiome in UC involves the reduction of conventional treatment pressure (especially, antibiotics [106–108]) on microbiota and growth promotion of “anti-inflammatory” bacteria. This approach could be further expanded to the preclinical drug approval process to ensure that our drugs in development have the lowest possible impact on the status of our normal commensals [107]. Not only will this aid in improving the outcomes of individual patients, but will also help with infection control efforts to prevent and/or shorten the duration of resistant bacteria colonization. One of the possibilities in this field is use of combined formulations, containing the conventional drug and prebiotic, as discussed below.

Gut microbiota markers for UC

Several specific bacteria that are associated with UC are described above in the section Gut dysbiosis is a risk factor for the UC. Analytic strategies to identify microbiota relevant to disease risk or disease activity in individual UC patients were described recently [109]. A study of colonic crypt mucus in patients with UC found the positive correlation between crypt bacterial load and subacute disease activity in UC, whereas bacterial load was reduced in acute UC [110]. Kolho et al demonstrated that in children with IBD intensity of intestinal inflammation was positively associated with reduced microbial diversity, abundance of butyrate producers, and relative abundance of Clostridium clusters IV and XIVa [111]. Quantitative variations in different species of Lactobacillus have been found between active UC and remission [77]. A consistent decrease of F. prausnitzii have been demonstrated in UC remission [96]; most prominent decrease in the concentration of F. prausnitzii was associated with a four-fold increase in the risk of relapse. However, inter-individual variations of microbiota are larger than inter-disease differences [63, 112], which makes the latter difficult to use as a diagnostic or prognostic tool. Moreover, biopsy locations less than 1 cm apart are different in composition of microbiota, thus limiting the diagnostic power of microbiota assessment [113].

Some attempts have been made to use microbiota as a biomarker. Firmicutes have been reported to be increased in UC patients who responded to mesalazine [100]. During the induction therapy with anti–TNF-α, the microbial diversity and similarity to the microbiota of healthy controls increased in the responder group by week 6 but not in the non-responders [105]. The abundance of 6 groups of bacteria, including those related to Eubacterium rectale and Bifidobacterium spp., predicted the response to anti–TNF-α medication [111]. Firmicutes:Bacteroidetes ratio shift has been proposed as a predictor of clinical outcome in IBD [114]. Microbiota metabolism, namely SCFA levels have been reported to monitor the response to prebiotic treatment [115].

Targeting microbiota: role of prebiotics

Increasing data on the human microbiota involvement in intestinal inflammation has led to investigation of the potentially therapeutic effect of prebiotics in IBD. Prebiotics are generally regarded as non-digestible food ingredients that are fermented by intestinal bacteria in a selective manner which promote changes in the gut ecosystem that benefit the host [116]. No formal definition of prebiotics is established, though [117, 118]. It is required, however, that to be characterised as a prebiotic, a substance should meet the following criteria: 1. The fermentability should be demonstrated in in vitro tests that simulate, for example, physiological conditions found in the gut. Promising substrates should be evaluated in randomised and placebo-controlled clinical studies, in order to confirm the positive outcomes obtained by in vitro studies; 2. The main trait of a prebiotic is to be a selective substrate for one or more beneficial gut commensal bacteria, which are stimulated to multiply and/or are metabolically activated, beneficially altering the colonic microbiota composition of the host. To confirm the selectivity of a prebiotic, it is of great importance to monitor the changes in the faecal microbiota during supplementation studies with the prebiotic through in vitro and in vivo tests. Moreover, selectivity consists of a key attribute that distinguishes prebiotics from other dietary fibres [119].

Prebiotics include oligosaccharides, which further divide into fructo-oligosaccharides (FOS) (oligofructose and inulin which have been shown to increase commensal anti-inflammatory faecal and mucosal Bifidobacteria and Faecalibacterium prausnitzii in healthy humans [120]), galacto-oligosaccharides (lactulose), and gluco- and xylo-oligosaccharides, psyllium (Plantago ovata seeds); and germinated barley foodstuff (GBF). Prebiotic fermentation results in the production of SCFA and gas (CO2 + H2) [121]. The subsequent drop in pH favors an increase in Bifidobacteria, Lactobacilli and non-pathogenic E. coli, while decreasing Bacteroidaceae and inhibition of some strains of pathogenic bacteria, e.g., Clostridium spp [121]. This fermentation of carbohydrates also leads to the production of butyrate acids, which has been proven to exert anti-inflammatory action. One of the proposed mechanisms of beneficial prebiotic action on the mucosa is a decreased activity of the endocannabinoid system (ECS) in the gut and an increased level of glucagon-like peptide-2, which stimulates tight junctions formation [122].

Only a small number of clinical trials have assessed the use of prebiotics in UC. Despite convincing and reproducible results from animal studies showing multiple benefits in IBD (e.g. [123–126], the data in humans remain scarce and not so encouraging. There is an issue with the dosage regimens of prebiotics. Typical efficacious dose in animal studies is 10% w/w of the diet, which in humans equates to about 50 g/day [13]. Lactulose, for example, provides positive effects starting from 10 g/day, which is considered as a low dose; usual regimens fall into the range of 30-60 g/day, producing laxative effect [127–132] and lowering the dose is considered to avoid this while preserving the beneficial prebiotic properties [115]. In one small non-controlled study, lactulose use provided no clinical or endoscopic improvement in UC [133]. However, UC patients showed an improvement in the quality of life.

The high water-holding capacity of the prebiotic germinated barley foodstuff (GBF) has been found to help modulate stool water content, leading to amelioration of symptoms in IBD [134]. In induction of remission, GBF had a significant decrease in clinical ± endoscopic activity in 3 open-label trials of the same team [134–137]. Kanauchi et al. found that 4 weeks of GBF in UC patients results in both clinical and endoscopic improvement. Exacerbation of disease with completion of GBF suggests that long-term or chronic treatment might be required for this therapy to be permanently effective. With regards to its effect on the human microbiome, GBF administration has led to an increased abundance of luminal Bifidobacterium and Eubacterium, as well as increased butyrate levels, resulting in attenuation of colitis.

In a small RCT (n=19), FOS+inulin vs placebo, added in both groups to mesalamine, had a significant decrease in fecal calprotectin level, but not in disease activity, possibly due to short-term study period of 2 weeks [138]. Another prebiotic, bifidogenic growth stimulator led, in an open-label trial (n=12), to a significant decrease in clinical and endoscopic activity in UC patients [139].

For maintenance of remission, the largest RCT (n=102) was conducted where prebiotic was found to be non-inferior to mesalamine [140]. GBF added to medication in an open-label trial was significantly superior to medication only in reducing the recurrence rate [136].

A head-to-head trial comparing the efficacy of probiotic, prebiotic, and synbiotic in UC [141] showed a superior benefit in quality of life with the use of symbiotic compared to probiotic or prebiotic alone. Another study showed no difference between synbiotic (Bifidobacterium longum+inulin/oligofructose 6 g/d) and placebo in terms of clinical disease activity. However, a significant decrease in colonic endoscopic signs was observed in the synbiotic group compared to placebo. Inflammation markers in the mucosa (TNFα, IL-1, β‐defensins decreased in the group receiving synbiotic [142].

Two studies have examined the use of pre- and synbotics in pouchitis (a common complication in patients undergoing restorative proctocolectomy for UC). In an open-label study [143], 10 patients with antibiotic-refractory or antibiotic-dependent pouchitis were treated with combination of Lactobacillus GG and FOS. Complete clinical and endoscopic remission have been achieved one month after the treatment start. In a double-blind crossover trial by Welters et al. [144], 20 UC and FAP patients with subclinical chronic pouchitis were randomized to receive dietary inulin 24 g/d or placebo for two 3-week study phases interconnected by 4-week washout period. Although patients with pouchitis were not specifically excluded, none of the enrolled patients had overtly clinical pouchitis. No change in clinical activity scores was detected during the inulin period, although there was an endoscopic and histological reversal of mucosal inflammation, reflected by a significant reduction of pouchitis disease activity index. This drives us to the nest section, where combination of known drugs and prebiotics are discussed.

While these findings are overall promising, there is currently no substantial evidence arguing for or against prebiotics in the treatment of IBD, and they continue to be used as a supplement to conventional IBD therapy rather than as a replacement. Furthermore, data on the effect they have on the microflora remain limited.

There are anecdotal studies on the use of prebiotics in combination with anti-colitis treatments. Schulz et al [145] tested the efficacy of a prebiotic/probiotic formulation (Lactobacillus acidophilus, Bifidobacterium lactis + inulin) in combination with metronidazole in rat model of colitis. Probiotic/prebiotic combination alone reduced inflammation while no added benefit has been found in combination with metronidazole. Interestingly, the authors make a note that anti-inflammatory effect has been persuaded by prebiotic.

Clearly, studies on combination of prebiotics and traditional drugs used in UC treatment are lacking. However, we can speculate that possible combinations not investigated yet in treament of UC may include:

Oral 5-ASA + prebiotic

Oral antimicrobials + prebiotic

Oral GCS + prebiotic

One of the studies demonstrated that antibiotics suppress anti-inflammatory effects of lactulose and authors declare that combined antibiotic/lactulose administration is not indicated [146]. However, lactulose still prevented the colon from shortening; furthermore, in a small study combined lactulose+antibiotics dosage forms have proved to be effective in terms of preservation of normal microbiota during antibiotic treatment [115]. Combined formulations of 5-ASA and prebiotics seem to be a reasonable option since concurrent lactulose administration doesn’t seem to affect oral 5-ASA pharmacokinetics despite luminal content acidification [147]. Moreover, lactulose-containing formulations can be used for colon-targeted drug delivery which is crucial in the case of both 5-ASA and topical GCSs [148]. Powder for oral suspension containing drug-loaded microspheres is a convenient formulation, easily transportable and allowing accurate dosage as well as use in persons with swallowing difficulties. Granules also can mask an unpleasant taste, thus increasing compliance.

In conclusion, use of prebiotics is an encouraging treatment modality for UC; design of novel drug formulations, containing both prebiotic and traditional drug is a promising strategy. More well-designed trials are needed to confirm the early results and to accelerate the development of treatment regimens using the prebiotics.